While it is now well established that actin plays widely conserved role in receptor-mediated endocytosis, actin's specific roles, and how actin polymerization is restricted to the vesicle budding stage of endocytosis, are poorly understood. The experiments proposed herein address actin's endocytic function in mammalian cells. Key questions to be addressed include: (1) How is actin assembly initiated at endocytic sites? (2) What functions are performed by actin at specific steps in the pathway? (3) How are actin-associated forces harnessed during endocytosis? (4) How are the activities of proteins that mediate actin's endocytic function regulated? Aim 1: To determine the biological roles and regulatory mechanisms for proteins at the actin-endocytic machinery interface. Hypotheses to be tested are (1) that actin assembly is nucleated around endocytic coats for development of deeply invaginated, constricted clathrin-coated pits and for vesicle scission, and (2) that a negative feedback mechanism turns off actin assembly shortly after it is initiated. Analysis of the dynamic behaviors, protein levels, stoichiometries and ultrastructural organization of endocytic proteins, and how functional perturbation of these proteins affects endocytosis, will test these hypotheses. Aim2: Proteins including dynamin, syndapin, intersectin, N-WASP, cortactin, the Arp2/3 complex, and Hip1 R are hypothesized to promote and regulate actin assembly at endocytic sites, and to harness the forces generated for invagination, constriction, and scission. Roles for these proteins will be tested using the lab's expertise in actin assembly biochemistry. Biochemical activities of the individual proteins will be determined, as will the emergent properties that result when different proteins are combined. A pyrene actin assembly assay, and reconstitution of assembly from liposome surfaces with the ultimate aim of driving actin-facilitated vesicle formation, will be used. Data from all studies will be synthesized for mathematical modeling of actin's role(s) in endocytic internalization. Public Health Relevance: The plasma membrane is critically important for cell growth regulation, nutrient absorption, and defense against pathogens and toxins. Endocytosis is important for these functions because of its role in control of plasma membrane protein and lipid composition, regulation of signaling pathways, control of cell surface area, and uptake of nutrients and pathogens. Endocytic defects have been linked to heart disease and cancer.